Determination and detection of nucleotide sequences and measurement of molecular weights regarding nucleic acids such as DNA and RNA, are the most important analytical means in biochemical and molecular biological research, and recently, have also become an important tool in gene diagnosis and gene therapy. Generally, in the case where these analytical means are applied to biological test samples such as blood, urine, cerebrospinal fluid, and saliva, the analysis is performed after amplifying nucleic acid molecules present in an extremely small amount in the test sample to an appropriate concentration, in order to increase accuracy in the analysis. For example, in PCR (polymerase chain reaction), which is a very important gene amplification method, two types of primers work together in a plurality of amplification cycles. An amplified gene through appropriate amplification cycles is finally detected by subjecting the amplified sample to gel electrophoresis to separate them into fractions based on chain length (molecular weight), and then, determining the presence and absence of a fraction corresponding to a specific chain length.
In the PCR, a target nucleic acid is treated in the presence of DNA polymerase serving as a nucleic acid synthase, primers for initiating replication, and four types of bases (dATP, dGTP, dTTP, dCTP) serving as substrates for use in replication, under conditions in which a polymerizing reaction of the substrate molecules can take place. It follows that a complementary chain is synthesized by using a single-stranded DNA derived from the target nucleic acid as a template to form a double stranded DNA (dsDNA), resulting in amplification of the nucleic acid. As described, the PCR product amplified by co-working of two types of primers always forms a double stranded DNA (dsDNA).
The hybridization reaction using an oligonucleotide probe having a specific nucleotide sequence is used for determining a nucleotide sequence of the PCR product, namely, dsDNA. However, the hybridization reaction using the oligonucleotide probe has such problems that non-specific hybridization easily takes place because quick annealing must be repeated, and further, both DNA chains resemble the probe in nucleotide sequence.
As a quantitative method for measuring a total amount of dsDNA after amplified by the PCR method, some methods have been proposed hitherto. For instance, one of the methods is as follows. PCR is effected in a predetermined number of cycles using a sufficient amount of a PCR primer labeled with a marker molecule such as a fluorescent dye. The PCR product obtained through the predetermined cycles is subjected to electrophoresis to separate into a primer elongated by polymerase reaction and a free primer not elongated (Bound/Free separation). After the bound/free separation, a total number of fluorescent molecules is counted by a fluorometer. Also known is another method using, as the fluorescent marker, an intercalator (e.g. acridine orange, thiazole orange, oxazole yellow, etc.) which cannot emit fluorescence until it intercalates into the dsDNA during the amplification process. In this method, it is possible to measure the intensity of fluorescent which is emitted from dsDNA hybridized in a test solution only, without effecting the Bound/Free separation.
In the methods described above which measure a total fluorescent amount obtained from a whole test solution, the primers are used in a sufficient amount so as to cause no shortage of the primer during the nucleic acid amplification process of predetermined cycles regardless of an initial concentration of a target nucleic acid molecule. However, in the amplification reaction in the PCR method, various reaction curves are obtained depending upon not only the number (or amount) but also types of target nucleic acid molecules present in a test sample. It is therefore difficult for a conventional analytic method, which measures a total amount of the marker molecules attached to the amplified nucleic acids, to quantitatively monitor the reaction process. In particular, in the conventional PCR method, a signal derived from the free labeled molecules is eliminated and only a signal from the labeled primer hybridized with the target nucleic acid is measured, by removing free labeled molecules through the Bound/Free separation or by employing the intercalator. It is therefore impossible to monitor whether a particular free primer molecule is hybridized or not. Accordingly, in the conventional target nucleic acid analysis, it is impossible to perform quantitative analysis in the presence of a free primer molecule. Furthermore, since a total amount of fluorescence of the whole test solution is measured, the amount of the test solution must be increased in order to improve the sensitivity of the measurement. In addition, since the measurement cannot be performed until a nucleic acid is sufficiently amplified, it is almost impossible to detect a target nucleic acid in an earlier stage of amplification. More specifically, it is difficult to perform quantitative analysis for determining the number (or amount) of the target nucleic acids in the mid-stage of the PCR amplification process, let alone in the earlier stage of the process.
On the other hand, a technique has been lately developed for accurately determining micro-motions of individual target molecules by measuring fluctuation motion of the target molecule in fluid and applying an autocorrelation function. The method of this type is called as a fluorescence correlation spectroscopy (referred to as “FCS” hereinafter) since a fluorescent marker molecule is measured by an optical instrument. An arithmetical operation using the FCS for processing data regarding a biological material are disclosed by Kinjo et al., in which FCS is employed in a hybridization reaction between a labeled nucleic acid probe and a target nucleic acid molecule (Kinjo, M., Rigler, R., Nucleic Acids Research, 23, 1795–1799, 1995).
As a method for detecting a target gene by use of FCS, some reports have been recently provided. According to the report made by Oehlenschlager, F. et al. (Proc. Natl. Acad. Sci. USA, 93, 12811–12816, 1996), it is demonstrated that NASBA (Nucleic Acid Sequence Based Amplification) combined with FCS is an effective detection method for diagnosing HIV (Human Immunodeficiency Virus) in a serum. The NASBA method, which is a modified PCR method, requires an additional primer labeled with fluorescence as a probe other than two primers described above in the presence of three enzymes, i.e., T7 RNA polymerase, reverse transcriptase, and RNaseH.
As a more simplified method than the NASBA method, an amplified probe extension (APEX) using FCS has been reported from the same group (Walter, N. G. et al., Proc. Natl. Acad. Sci. USA, 93, 12805–12810, 1996). Even in the simplified method, an additional primer labeled with fluorescence is also required as a probe, besides a set of a forward primer and a reverse primer. In an experiment of APEX, change in autocorrelation function value with time is observed after 26 cycles.
However, in both the NASBA method and the APEX method, since an additional labeled probe is required besides the materials for use in general nucleic acid amplification, reaction conditions becomes complicated, making it difficult to stably control an accuracy of analysis. Furthermore, since the probe molecule is labeled with a single fluorescent marker, a signal derived from a labeled free probe significantly reduces an S/N ratio. As a result, the detection sensitivity tends to decrease as the initial concentration of the target nucleic acid molecule decreases. Furthermore, many cycles of PCR reactions must be performed to ensure the detection.